Mycoplasma hominis strain 1620 was isolated from the synovial fluid of a chronically infected septic arthritis patient. We have been studying the attachment specificities of this pathogenic strain and find it adheres to sulfatide as well as gangliosides, without any apparent requirement for sialic acid. Cultures of M. hominis strain 1620 were grown in media containing 14C- palmitate; the labeled cells were used to overlay thin layer chromatograms containing 1 mg each of various glycolipids. This strain of M. hominis was observed to bind avidly to sulfatides and slightly to CDH, but not to GM1, asialoGM1, GD1a, GD1b, GT1b, GM2 and GM3, but not the neutral glycolipids, CTH, globoside or Forssman glycolipid. This observation leads us to believe the linkage of the sugar(s) may be important. Binding of M. hominis to sulfatide adsorbed to plastic of microtiter plate wells was used to demonstrate that the binding is temperature and dose dependent. The mycoplasma also binds to cholesterol sulfate in a dose and temperature- dependent fashion, but only at concentrations 100X those of sulfatide.